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Molecular Evidence for the Monophyly of Tenrecidae (Mammalia) and the Timing of the Colonization of Madagascar by Malagasy Tenrecs
Molecular Phylogenetics and Evolution Vol. 22, No. 3, March, pp. 357–363, 2002 doi:10.1006/mpev.2001.1055, available online at http://www.idealibrary.com on
Molecular Evidence for the Monophyly of Tenrecidae (Mammalia) and the Timing of the Colonization of Madagascar by Malagasy Tenrecs Christophe J. Douady,* Francois Catzeflis,† Diana J. Kao,‡ Mark S. Springer,‡ ,1 and Michael J. Stanhope* ,1,2 *Biology and Biochemistry, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom; †Laboratoire de Pale´ontologie, CC 064, I.S.E.M., UMR 5554 CNRS, Place E. Bataillon, F-34095 Montpellier, France; and ‡Department of Biology, University of California, Riverside, California 92521 Received January 18, 2001; revised July 18, 2001
INTRODUCTION Tenrecs are a diverse family of insectivores, with an Afro-Malagasian biogeographic distribution. Three subfamilies (Geogalinae, Oryzorictinae, Tenrecinae) are restricted to Madagascar and one subfamily, the otter shrews (Potamogalinae), occurs on the mainland. Morphological studies have generated conflicting hypotheses according to which both tenrecids and Malagassy tenrecs are either monophyletic or paraphyletic. Competing hypotheses have different implications for the biogeographic history of Tenrecidae. At present, there are no molecular studies that address these hypotheses. The present study provides sequences of a nuclear protein-coding gene (vWF) and the mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes from a potamogaline (Micropotamogale). New sequences of these genes are also reported for the tenrecine, Tenrec ecaudatus. The 12S sequences from these taxa were combined with data already available for this locus from two other tenrecids (Echinops telfairi, subfamily Tenrecinae and Oryzorictes talpoides, subfamily Oryzorictinae). Phylogenetic analyses provided strong bootstrap support for the monophyly of Tenrecidae and Malagasy tenrecs. The majority of statistical tests rejected morphological claims for both a Tenrecinae–Chrysochloridae clade and an Oryzorictinae–Potamogalinae clade. Molecular clock estimates suggest a split of otter shrews and Malagasy tenrecs at approximately 53 MYA. We estimate that the ancestor of Malagasy tenrecs dispersed to Madagascar subsequent to this split but prior to about 37 MYA. © 2002 Elsevier Science (USA)
1
To whom correspondence and reprint requests should be addressed. Fax: (610) 917-7901. E-mail: Michael_J_Stanhope@ gsk.com or [email protected]. 2 Current address: Bioinformatics, GlaxoSmithKline, 1250 South Collegeville Rd., Collegeville, PA 19426-0989.
The taxonomic wastebasket “Insectivora” remains one of the most misunderstood mammalian groups. Throughout the history of mammalian systematic study, the taxonomic composition of the group has been debated and modified (see, e.g., Van Valen, 1967; McKenna, 1975; Yates, 1984; McKenna and Bell, 1997). While the overall primitive condition of insectivores has undoubtedly contributed to this enigmatic status, presumed similarities between the insectivores and the ancestral placental stock (Simpson, 1945) have also made insectivores a group of special interest in the study of mammalian evolution. Among the six lipotyphlan insectivore families [Chrysochloridae (⫽ golden moles), Erinaceidae (⫽ hedgehogs and gymnures), Solenodontidae (⫽ solenodons), Soricidae (⫽ shrews), Talpidae (⫽ moles), and Tenrecidae (⫽ tenrecs)], the Afro-Malagasy tenrecs have traditionally been placed in the suborder Soricomorpha with shrews, moles, solenodons, and, more variably, golden moles (Butler, 1988; MacPhee and Novacek, 1993; but see Van Valen, 1967 and McKenna, 1975). In contrast, phylogenetic analyses with DNA sequences demonstrate that tenrecs are closely related to golden moles but not to other soricomorphs (Stanhope et al., 1998a,b). Further, tenrecs and golden moles share a common ancestry with five African-origin orders to the exclusion of other lipotyphlans (Springer et al., 1997; Stanhope et al., 1998a,b; Liu and Miyamoto, 1999; Madsen et al., 2001). Stanhope et al. (1998a) suggested the name “Afrosoricida” for a new order of mammals that includes tenrecids and chrysochlorids. Afrotheria (Stanhope et al., 1998a) is the superordinal group that includes “Afrosoricida,” Sirenia, Proboscidea, Hyracoidea, Macroscelidea, and Tubulidentata. Whereas some authors have argued for a tenrecid– chrysochlorid alliance based on morphology (e.g., Butler, 1988), the inclusion of “Afrosoricida” in Afrotheria is unprece-
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1055-7903/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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dented based on morphological data and contradicts more than one century of morphological investigation (e.g., Haeckel, 1866; Gregory, 1910; Butler, 1988; MacPhee and Novacek, 1993). Even the recent morphological study of Asher (1999), which fails to support lipotyphlan monophyly, finds no support for Afrotheria. The family Tenrecidae has conventionally been viewed as monophyletic (Butler, 1972). In contrast, four of eight analyses presented by Asher (1999) challenge the monophyly of Tenrecidae and instead suggest that a subfamily of tenrecs, Tenrecinae, are more closely related to chrysochlorids (golden moles) than to other tenrecs, creating a paraphyletic Tenrecidae. Asher’s analyses never recovered the monophyly of the Malagasy tenrecs and instead placed Limnogale (subfamily Oryzorictinae) as the sister taxon to the African otter shrews (subfamily Potamogalinae) in eight of eight analyses. Finally, Asher’s (1999) study supports the hypothesis that there have been multiple colonization events of Madagascar by tenrecs. At present, there are no published molecular studies that address questions pertaining to tenrecid monophyly, the monophyly of Malagasy tenrecs, and the biogeographic history of Tenrecidae. The present paper provides DNA sequences for a potamogaline and examines the evolutionary history of the Tenrecidae from a molecular perspective. MATERIALS AND METHODS Phylogenetic Loci and Taxa Given the suggestion that basal tenrecid divergences may trace to the Paleocene (Eisenberg, 1981), we selected moderately conserved loci that have phylogenetic signal at this temporal level (Springer et al., 2001). Specifically, we collected and analyzed DNA sequences for two independent data sets: exon 28 of the gene encoding von Willebrand Factor (vWF) and the colinear mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes. The data sets that we analyzed encompassed 36 taxa that represent all orders of placental mammals for both vWF and 12S–16S rRNA (Table 1; sequences indicated with an asterisk are new to this study). The Tenrecidae included in these analyses are Micropotamogale lamottei (Nimba Otter Shrew), Tenrec ecaudatus (Tailless Tenrec), and Echinops telfairi (Lesser Hedgehog Tenrec). The Micropotamogale and Tenrec vWF genes were amplified and sequenced as described previously (Porter et al., 1996; Springer et al., 1997). The 12S rRNA–tRNA valine–16S rRNA amplification primers for Micropotamogale were modified to correspond to “afrotherian versions” of primers 12C (Springer et al., 1995) and 16R (Springer et al., 1997). The sequences of these two primers are 5⬘ AAA GCA AAR CAC TGA AAA TGC YTA GAT G 3⬘ and 5⬘ TGT
TAA GGA GAG GAT TTG AAC CTC TG 3⬘, respectively. Mitochondrial RNA gene sequences of other mammals (Table 1) were obtained as previously described (Springer et al., 1997). The resulting data sets were aligned using Clustal X (Thompson et al., 1994) and subsequently refined by eye. Positions that could not be unambiguously aligned by eye were excluded from analysis. The secondary structures of the 12S and 16S rRNA molecules (Springer and Douzery, 1996; Burk, 1999) allowed some further refinement of the final alignment. The resulting vWF alignment was 1254 bp in length and the 12S rRNA–tRNA valine–16S rRNA alignment was 2053 bp in length. Both alignments are available upon request from M.J.S. ([email protected]). Finally, we analyzed a matrix of 12S rRNA gene sequences that also included the Oryzorictes talpoides sequence from Emerson et al. (1999). Oryzorictes was added manually to the previously described rRNA alignment. Analysis Phylogenetic reconstructions. Maximum-likelihood (ML), minimum-evolution (ME), and maximum-parsimony (MP) were used to infer phylogenetic relationships. The majority of these analyses were carried out using PAUP*4.0b2 (Swofford, 1998); Puzzle 4.0.2 (Strimmer and von Haeseler, 1996) was used to perform quartet puzzling (QP) with vWF amino acid sequences. ML and QP analyses employed the Tamura and Nei model (Tamura and Nei, 1993). For each data set, ML and QP searches were performed with and without a gamma distribution (G) of rates across sites and an allowance for a proportion of invariant sites (I). Substitution rates, the shape parameter of the gamma distribution, and the proportion of invariant sites were estimated from the most parsimonious trees for the corresponding data or, in the case of QP, directly from the data set. ME trees were computed using LogDet (Lockhart et al., 1994) and GTR (General Time Reversible; Lanave et al., 1984) distances. In all analyses, gaps where treated as missing data, branches were swapped using the tree bisection and reconnection branch swapping option, and the taxon addition sequence was random. The statistical significance of a priori hypotheses was evaluated with the Winning Sites (WS; Prager and Wilson, 1988), Templeton (T; 1983), and Kishino-Hasegawa (KH; 1989) tests. These calculations were also done using PAUP*4.0b2. Clade support was assessed using the bootstrap, employing 200 replicates for ML, 500 replicates for ME and MP, and 10,000 puzzling steps for QP. Trees were rooted using marsupial outgroups. Molecular clock estimates of splitting events. The relative evolutionary rate homogeneity between taxa was tested using the method proposed by Wu and Li
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TABLE 1 Accession Numbers and Taxa Common Names Species
Common name
vWF Accession No.
12S-16S Accession No.
Didelphis virginiana Macropus giganteus Bradypus tridactylus Chaetophractus villosus Erinaceus europaeus Scalopus aquaticus Tupaia (glis/tana) Cynocephalus variegatus Megaderma lyra Tadarida brasiliensis Cynopterus sphinx Dobsonia moluccensis Otolemur crassicaudatus Homo sapiens Felis catus Canis familiaris Phocoena phocoena Balaenoptera physalus Sus scrofa Bos taurus Equus (asinus/caballus) Ceratotherium simum Manis sp. Mus domesticus Cavia porcellus Dasyprocta agouti Oryctolagus cuniculus Ochotona princeps Dugong dugon Loxodonta africana Elephas maximus Procavia capensis Orycteropus afer Elephantulus rufescens Amblysomus hottentotus Tenrec ecaudatus Echinops telfairi Micropotamogale lamottei
Opossum Kangaroo Sloth Armadillo Hedgehog Mole Tree shrew Flying lemur Microbat Microbat Megabat Megabat Galago Human Cat Dog Porpoise Fin whale Pig Cow Horse Rhino Pangolin Mouse Guinea pig Agouti Rabbit Pika Dugong African elephant Asian elephant Hyrax Aardvark Elephant shrew Golden mole Tailless tenrec Lesser hedgehog tenrec Nimba otter shrew
AF226848 AJ224670 U31603 AF076480 U97536 AF076479 U31623, AF061063 U31606 U31616 U31623, AF061061 U31605 U31609 U31614, AFO61064 M25851 U31613, AFO61O62 L76227 AF061060 — S78431 X63820, AF004285 U31610 U31604 U97535 U27810 — U31607 U31618 AJ224672 U31608 U31615 U31611 U31619 U31617 U31612 U97534 AF390536* AF076478 AF390538
Z29573 AF027985 AF069535, AF038022 U61080, AF069534 X88898 AF069539 AF038021, AF203727 AF038018, AF038018 AF069538 AF179288 U93068, AF203740 U93065, AF179290 AF019080 J01415 U20753 U96639 — X61145 AJ002189 J01393 X79547 Y07726 U97340, U61079 J01420 L35585 — AJ001588 AF390540* U60185, AF179291 U60182, AF039436 AF390541* U97335, U60184 U97338 U97339 U97336 AF390537* AF069540 AF390539*
Note. Afrotheria members appear underlined.
(1985), as developed in the program K2WuLi-1.0 (Jermiin, 1996), and by the Unambiguous Parsimony Sites test (e.g., Mindell and Honeycut, 1990; Waddell et al., 1999). Taxa showing rate heterogeneity were removed from the analysis (taxa retained for each of the loci were as follows: Vwf: Macropus, Bos, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops; 12S: Macropus, Manis, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong; 16S: Cyclopes, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong; 12S–16S: Balaenoptera, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong). The resulting pruned taxon set was then analyzed by ML (TN model with parameters estimated as above) under a clock constraint.
We used basal divergences among paenungulate orders (i.e., a hyrax– elephant split, chosen because vWF dugong was not clocklike) at 60 million years ago (MYA) (Amrine and Springer, 1999) as a calibration point for estimating cladogenic events within Tenrecidae. An estimate of variation on these dates was obtained by calculating the split using four different data sets: vWF, 12S rRNA, 16S rRNA, and a concatenation of 12S and 16S rRNA. RESULTS AND DISCUSSION Tenrecidae Monophyly Both the 12S–16S rRNA and the vWF data sets resulted in high support for the monophyly of Tenrecidae (Fig. 1). For the vWF locus, support values (boot-
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FIG. 1. Maximum-likelihood trees based on vWF (A) and 12S–16S (B) data sets. Topologies and branch lengths were determined under a TN model assuming rate variation across sites and employing a proportion of invariant sites.
strap and puzzle) for this clade ranged from 77 to 100%, with only the amino acid puzzle support value below 94% (Table 2). For 12S–16S rRNA, bootstrap support ranged from 80 to 95% (Table 2). Combining the two sets of data into a single concatenated alignment resulted in 100% bootstrap support for the monophyly of Tenrecidae. With the exception of KH-ML for vWF, statistical tests involving a single locus did not provide significant support for Tenrecidae monophyly (Table 2). However, the concatenated data set did provide significant support for tenrecid monophyly, both with MP and ML (Table 2). Asher’s (1999) hypothesis of a paraphyletic Tenrecidae, wherein tenrecines are more closely related to golden moles than to other tenrecids, was rejected with the vWF and concatenated data sets (Table 2). Although statistical tests with the 12S–16S rRNA data set did not reject the Asher hypothesis, bootstrap support was minimal and ranged from 3 to 10%. All of Asher’s (1999) analyses recovered a sister
group relationship between Limnogale and potamogalines, which implies paraphyly of Malagasy tenrecs. This conclusion was mainly supported by a close Potamogalinae–Limnogale (subfamily Oryzorictinae) relationship, but also by an association of the remaining Oryzorictinae with this clade to the exclusion of tenrecines. The recent publication of a 12S rRNA gene sequence for Oryzorictes (Emerson et al., 1999) allowed us to examine the naturalness of a Potamogalinae– Oryzorictinae grouping. After addition of this taxon to our 12S sequences (same selection of taxa as the 12S– 16S analyses), MP, ME, and ML 12S rRNA bootstrap trees supported the monophyly of Tenrecidae (bootstrap support ranged from 47 to 71%) and an Oryzorictes– Tenrecinae clade (bootstrap support ranged from 65 to 80%). Data sets of 12S rRNA sequences confined to afrotherian species (Table 1), with two different sets of outgroups (two Xenarthra or two Chiroptera), recovered additional support for this Oryzorictes– Tenrecinae clade (Table 3). All methods of phylogenetic
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TABLE 2 Tenrecidae Monophyly versus Asher’s Paraphyletic (Tenrecinae ⴙ Chrysochloridae) Hypothesis Tenrecinae ⫹ Golden mole
Tenrecidae
Bootstrap DNA ME (GTR) (Logdet) MP ML (TN) ML (TN GI) Protein MP QP (Blosum 62) QP (Blosum 62 GI) Decay and statistical tests DNA MP Decay KH T WS ML (TN) KH ML (TN GI) KH Protein MP Decay KH T WS
12S–16S
vWF
Concat.
12S–16S
vWF
Concat.
94 95 80 85 93
100 100 99 94 95
100 100 100 100 100
5 3 8 10 6
0 0 0 1 0
0 0 0 0 0
NA NA NA
99 77 77
NA NA NA
NA NA NA
0 ⬍50 ⬍50
NA NA NA
8 0.588–0.634 0.495–0.520 0.402–0.545 0.399 0.204
19 0.189 0.223 0.292 0.000 0.290
31 0.026 0.025 0.049 0.000 0.019
⫺8 0.588–0.634 0.495–0.520 0.402–0.545 0.509 0.538
⫺22 0.046–0.050 0.049–0.052 0.024–0.032 0.005 0.015
⫺38 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.000 0.019
NA NA NA NA
9 0.061 0.061 0.093
NA NA NA NA
NA NA NA NA
⫺13 0.007–0.028 0.007–0.028 0.011–0.043
NA NA NA NA
Note. Statistical test results for Tenrecidae correspond to acceptance of that hypothesis, whereas test results for Tenrecinae ⫹ Golden mole, refer to rejection.
reconstruction, regardless of the root, recovered this association with bootstrap support ranging from 86 to 100%. Bootstrap support for Oryzorictes–Micropotamogale was ⬍1% in all analyses and a subset of statistical tests rejected this hypothesis (Table 3).
Timing and Biogeographic Events The basal split between Potamogalinae and Tenrecinae was estimated at 53, 52, 53, and 51 MYA with vWF, 12S–16S, 12S, and 16S, respectively. Estimates
TABLE 3 Malagasy Tenrecid Monophyly versus Asher’s Paraphyletic (Potamogalinae–Oryzorictinae) Hypothesis Oryzorictes ⫹ Tenrecinae
Bootstrap ME (GTR) (Logdet) MP ML (TN) ML (TN GI) Decay and statistical tests MP Decay KH T WS ML (TN) ML (TN GI)
Oryzorictes ⫹ Micropotamogale
Xenarthra
Chiroptera
Xenarthra
Chiroptera
96 96 86 99 99
97 98 91 99 100
0 0 0 0 0
0 0 0 0 0
⫹6 0.4605 0.4602 0.2076 0.2665 0.0371
⫹6 0.2280 0.2278 0.2913 0.0548 0.0995
⫺9 0.2078 0.6143 0.2626 0.1314 0.0379
⫺12 0.0072–0.0640 0.0073–0.0641 0.0118–0.0896 0.0340 0.0995
Note. Statistical test results for Oryzorictes ⫹ Tenrecinae correspond to acceptance of that hypothesis, whereas test results for Oryzorictes ⫹ Micropotamogale, refer to rejection.
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for the Tenrec–Echinops split within Tenrecinae were more variable (18, 38, 30, 44 MYA). Adding Oryzorictes to the 12S data set resulted in a similar estimate for the split between Potamogalinae and other tenrecs at 55 MYA and placed the Oryzorictinae–Tenrecinae split at approximately 37 MYA. These dates are much older than the fossil record would suggest (i.e., the oldest tenrecids are Miocene; McKenna and Bell, 1997). The separation of Madagascar from mainland Africa occurred between 120 and 165 MYA (Rabinowitz et al., 1983), well before the origin of tenrecids. One or more dispersal events between Africa and Madagascar are thus required to explain the biogeographic history of tenrecids. Given that molecular data are compatible with both tenrecid monophyly and the monophyly of Malagasy tenrecs, the minimal requirement is a single dispersal event from Africa to Madagascar. Further, this event would have occurred after the potamogaline–Malagasy tenrec divergence (51 to 55 MYA) but before the radiation of Malagasy tenrecs (37 MYA based on the 12S rRNA data set that includes tenrecine and oryorictine representation). The mechanism by which tenrecids arrived in Madagascar remains unclear. McCall (1997) argued for a land bridge across the Mozambique Channel. According to this hypothesis, uplift along the Davie fracture zone created a filter bridge by which some terrestrial mammals were able to cross a deep channel with difficult currents, some time between the mid Eocene and the late Oligocene (45 to 26 MYA). The duration of the land bridge thus exhibits partial temporal overlap with the tenrecid dispersal window suggested by our molecular data. Although molecular phylogenies only require a single dispersal event for living tenrecids, fossil data suggest the possibility of a more complex dispersal history. Specifically, the subfamily Geogalinae includes the living Geogale (Madagascar) and the extinct genus Parageogale from the early Miocene of Africa (McKenna and Bell, 1997). Butler (1985) proposed a sister taxon relationship between Geogale and Parageogale. If these taxa are indeed close relatives, then a second dispersal event is required to account for the biogeographic history of the Geogalinae. Depending on the relationship of Geogalinae to other tenrecids, the second dispersal may have been from Africa to Madagascar or vice versa. At present, molecular data do not address this hypothesis. ACKNOWLEDGMENTS Otter shrew (Micropotamogale lamottei) biological material was a generous gift from Peter Vogel (IZEA—University of Lausanne, Switzerland), from a wild-caught specimen from Ye´ ale´ , Ivory Coast (voucher IZEA-7083). Peter Vogel also provided useful comments on an earlier version of the manuscript. This work was supported by grants from the Training and Mobility of Researchers program of the European Commission (ERB-FMRX-CT98-0221) to M.J.S. and the National Science Foundation (DEB-9903810) to M.S.S.
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TENREC PHYLOGENY AND BIOGEOGRAPHY Mindell, D. P., and Honeycutt, R. (1990). Ribosomal RNA in vertebrates: Evolution and phylogenetic applications. Annu. Rev. Ecol. Syst. 21: 541–566. Porter, C. A., Goodman, M., and Stanhope, M. J. (1996). Evidence on mammalian phylogeny from sequences of exon 28 of the von Willebrand factor gene. Mol. Phylogenet. Evol. 5: 89 –101. Prager, E. M., and Wilson, A. C. (1988). Ancient origin of lactalbumin from lysozyme: Analysis of DNA and amino acid sequences. J. Mol. Evol. 27: 326 –335. Rabinowitz, P. D., Coffin, M. F., and Falvey, D. (1983). The separation of Madagascar and Africa. Science 220: 67– 69. Simpson, G. G. (1945). The principles of classification and a classification of the mammals. Bull. Am. Mus. Nat. Hist. 85: 1–350. Springer, M. S., Hollar, L. J., and Burk, A. (1995). Compensatory substitutions and the evolution of the mitochondrial 12S rRNA gene in Mammals. Mol. Biol. Evol. 12: 1138 –1150. Springer, M. S., and Douzery, E. (1996). Secondary structure and patterns of evolution among mammalian mitochondrial 12S rRNA molecules. J. Mol. Evol. 43: 357–373. Springer, M. S., Cleven, G. C., Madsen, O., de Jong, W. W., Waddell, V. G., Amrine, H. M., and Stanhope, M. J. (1997). Endemic African mammals shake the phylogenetic tree. Nature 388: 61– 64. Springer, M. S., DeBry, R. W., Douady, C. J., Amrine, H. M., Madsen, O., de Jong, W. W., and Stanhope, M. J. (2001). Mitochondrial versus nuclear gene sequences in deep level mammalian phylogeny reconstruction. Mol. Biol. Evol. 18: 132–143. Stanhope, M. J., Waddell, V. G., Madsen, O., de Jong, W. W., Hedges, S. B., Cleven, G. C., Kao, D., and Springer, M. S. (1998a). Molecular evidence for multiple origins of Insectivora and for a new order of endemic African insectivore mammals. Proc. Natl. Acad. Sci. USA 95: 9967–9972. Stanhope, M. J., Madsen, O., Waddell, V. G., Cleven, G. C., de Jong,
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W. W., and Springer, M. S. (1998b). Highly congruent molecular support for a diverse superordinal clade of endemic African Mammals. Mol. Phylogenet. Evol. 9: 501–508. Strimmer, K., and von Haeseler, A. (1996). Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13: 964 –969. Swofford, D. L. (1998). PAUP*: Phylogenetic analysis using parsimony (* and other methods). Version 4. Sinauer, Sunderland, MA. Tamura, K., and Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512–526. Templeton, A. R. (1983). Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and apes. Evolution 37: 221–244. Thompson, J. D., Higgins, G. D., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positive specific gap penalties and weight matrix choice. Nucleic Acids. Res. 22: 4673– 4680. Van Valen, L. (1967). New Paleocene insectivores and insectivore classification. Bull. Am. Mus. Nat. Hist. 135: 217–284. Waddell, P. J., Cao, Y., Hasegawa, M., and Mindell, D. P. (1999). Assessing the Cretaceous superordinal divergence times within birds and placental mammals by using whole mitochondrial protein sequences and an extended statistical framework. Syst. Biol. 48: 119 –137. Wu, C.-I., and Li, W.-H. (1985). Evidence for higher rates of nucleotide substitution in rodents than in man. Proc. Natl. Acad. Sci. USA 82: 1741–1745. Yates, T. L. (1984). Insectivores, elephant shrews, tree shrews, and dermopterans. In “Orders and Families of Recent Mammals of the World” (S. Anderson and J. K. Jones, Jr., Eds.), pp. 117–144. Wiley, New York.
Molecular Evidence for the Monophyly of Tenrecidae (Mammalia) and the Timing of the Colonization of Madagascar by Malagasy Tenrecs Christophe J. Douady,* Francois Catzeflis,† Diana J. Kao,‡ Mark S. Springer,‡ ,1 and Michael J. Stanhope* ,1,2 *Biology and Biochemistry, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom; †Laboratoire de Pale´ontologie, CC 064, I.S.E.M., UMR 5554 CNRS, Place E. Bataillon, F-34095 Montpellier, France; and ‡Department of Biology, University of California, Riverside, California 92521 Received January 18, 2001; revised July 18, 2001
INTRODUCTION Tenrecs are a diverse family of insectivores, with an Afro-Malagasian biogeographic distribution. Three subfamilies (Geogalinae, Oryzorictinae, Tenrecinae) are restricted to Madagascar and one subfamily, the otter shrews (Potamogalinae), occurs on the mainland. Morphological studies have generated conflicting hypotheses according to which both tenrecids and Malagassy tenrecs are either monophyletic or paraphyletic. Competing hypotheses have different implications for the biogeographic history of Tenrecidae. At present, there are no molecular studies that address these hypotheses. The present study provides sequences of a nuclear protein-coding gene (vWF) and the mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes from a potamogaline (Micropotamogale). New sequences of these genes are also reported for the tenrecine, Tenrec ecaudatus. The 12S sequences from these taxa were combined with data already available for this locus from two other tenrecids (Echinops telfairi, subfamily Tenrecinae and Oryzorictes talpoides, subfamily Oryzorictinae). Phylogenetic analyses provided strong bootstrap support for the monophyly of Tenrecidae and Malagasy tenrecs. The majority of statistical tests rejected morphological claims for both a Tenrecinae–Chrysochloridae clade and an Oryzorictinae–Potamogalinae clade. Molecular clock estimates suggest a split of otter shrews and Malagasy tenrecs at approximately 53 MYA. We estimate that the ancestor of Malagasy tenrecs dispersed to Madagascar subsequent to this split but prior to about 37 MYA. © 2002 Elsevier Science (USA)
1
To whom correspondence and reprint requests should be addressed. Fax: (610) 917-7901. E-mail: Michael_J_Stanhope@ gsk.com or [email protected]. 2 Current address: Bioinformatics, GlaxoSmithKline, 1250 South Collegeville Rd., Collegeville, PA 19426-0989.
The taxonomic wastebasket “Insectivora” remains one of the most misunderstood mammalian groups. Throughout the history of mammalian systematic study, the taxonomic composition of the group has been debated and modified (see, e.g., Van Valen, 1967; McKenna, 1975; Yates, 1984; McKenna and Bell, 1997). While the overall primitive condition of insectivores has undoubtedly contributed to this enigmatic status, presumed similarities between the insectivores and the ancestral placental stock (Simpson, 1945) have also made insectivores a group of special interest in the study of mammalian evolution. Among the six lipotyphlan insectivore families [Chrysochloridae (⫽ golden moles), Erinaceidae (⫽ hedgehogs and gymnures), Solenodontidae (⫽ solenodons), Soricidae (⫽ shrews), Talpidae (⫽ moles), and Tenrecidae (⫽ tenrecs)], the Afro-Malagasy tenrecs have traditionally been placed in the suborder Soricomorpha with shrews, moles, solenodons, and, more variably, golden moles (Butler, 1988; MacPhee and Novacek, 1993; but see Van Valen, 1967 and McKenna, 1975). In contrast, phylogenetic analyses with DNA sequences demonstrate that tenrecs are closely related to golden moles but not to other soricomorphs (Stanhope et al., 1998a,b). Further, tenrecs and golden moles share a common ancestry with five African-origin orders to the exclusion of other lipotyphlans (Springer et al., 1997; Stanhope et al., 1998a,b; Liu and Miyamoto, 1999; Madsen et al., 2001). Stanhope et al. (1998a) suggested the name “Afrosoricida” for a new order of mammals that includes tenrecids and chrysochlorids. Afrotheria (Stanhope et al., 1998a) is the superordinal group that includes “Afrosoricida,” Sirenia, Proboscidea, Hyracoidea, Macroscelidea, and Tubulidentata. Whereas some authors have argued for a tenrecid– chrysochlorid alliance based on morphology (e.g., Butler, 1988), the inclusion of “Afrosoricida” in Afrotheria is unprece-
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1055-7903/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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dented based on morphological data and contradicts more than one century of morphological investigation (e.g., Haeckel, 1866; Gregory, 1910; Butler, 1988; MacPhee and Novacek, 1993). Even the recent morphological study of Asher (1999), which fails to support lipotyphlan monophyly, finds no support for Afrotheria. The family Tenrecidae has conventionally been viewed as monophyletic (Butler, 1972). In contrast, four of eight analyses presented by Asher (1999) challenge the monophyly of Tenrecidae and instead suggest that a subfamily of tenrecs, Tenrecinae, are more closely related to chrysochlorids (golden moles) than to other tenrecs, creating a paraphyletic Tenrecidae. Asher’s analyses never recovered the monophyly of the Malagasy tenrecs and instead placed Limnogale (subfamily Oryzorictinae) as the sister taxon to the African otter shrews (subfamily Potamogalinae) in eight of eight analyses. Finally, Asher’s (1999) study supports the hypothesis that there have been multiple colonization events of Madagascar by tenrecs. At present, there are no published molecular studies that address questions pertaining to tenrecid monophyly, the monophyly of Malagasy tenrecs, and the biogeographic history of Tenrecidae. The present paper provides DNA sequences for a potamogaline and examines the evolutionary history of the Tenrecidae from a molecular perspective. MATERIALS AND METHODS Phylogenetic Loci and Taxa Given the suggestion that basal tenrecid divergences may trace to the Paleocene (Eisenberg, 1981), we selected moderately conserved loci that have phylogenetic signal at this temporal level (Springer et al., 2001). Specifically, we collected and analyzed DNA sequences for two independent data sets: exon 28 of the gene encoding von Willebrand Factor (vWF) and the colinear mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes. The data sets that we analyzed encompassed 36 taxa that represent all orders of placental mammals for both vWF and 12S–16S rRNA (Table 1; sequences indicated with an asterisk are new to this study). The Tenrecidae included in these analyses are Micropotamogale lamottei (Nimba Otter Shrew), Tenrec ecaudatus (Tailless Tenrec), and Echinops telfairi (Lesser Hedgehog Tenrec). The Micropotamogale and Tenrec vWF genes were amplified and sequenced as described previously (Porter et al., 1996; Springer et al., 1997). The 12S rRNA–tRNA valine–16S rRNA amplification primers for Micropotamogale were modified to correspond to “afrotherian versions” of primers 12C (Springer et al., 1995) and 16R (Springer et al., 1997). The sequences of these two primers are 5⬘ AAA GCA AAR CAC TGA AAA TGC YTA GAT G 3⬘ and 5⬘ TGT
TAA GGA GAG GAT TTG AAC CTC TG 3⬘, respectively. Mitochondrial RNA gene sequences of other mammals (Table 1) were obtained as previously described (Springer et al., 1997). The resulting data sets were aligned using Clustal X (Thompson et al., 1994) and subsequently refined by eye. Positions that could not be unambiguously aligned by eye were excluded from analysis. The secondary structures of the 12S and 16S rRNA molecules (Springer and Douzery, 1996; Burk, 1999) allowed some further refinement of the final alignment. The resulting vWF alignment was 1254 bp in length and the 12S rRNA–tRNA valine–16S rRNA alignment was 2053 bp in length. Both alignments are available upon request from M.J.S. ([email protected]). Finally, we analyzed a matrix of 12S rRNA gene sequences that also included the Oryzorictes talpoides sequence from Emerson et al. (1999). Oryzorictes was added manually to the previously described rRNA alignment. Analysis Phylogenetic reconstructions. Maximum-likelihood (ML), minimum-evolution (ME), and maximum-parsimony (MP) were used to infer phylogenetic relationships. The majority of these analyses were carried out using PAUP*4.0b2 (Swofford, 1998); Puzzle 4.0.2 (Strimmer and von Haeseler, 1996) was used to perform quartet puzzling (QP) with vWF amino acid sequences. ML and QP analyses employed the Tamura and Nei model (Tamura and Nei, 1993). For each data set, ML and QP searches were performed with and without a gamma distribution (G) of rates across sites and an allowance for a proportion of invariant sites (I). Substitution rates, the shape parameter of the gamma distribution, and the proportion of invariant sites were estimated from the most parsimonious trees for the corresponding data or, in the case of QP, directly from the data set. ME trees were computed using LogDet (Lockhart et al., 1994) and GTR (General Time Reversible; Lanave et al., 1984) distances. In all analyses, gaps where treated as missing data, branches were swapped using the tree bisection and reconnection branch swapping option, and the taxon addition sequence was random. The statistical significance of a priori hypotheses was evaluated with the Winning Sites (WS; Prager and Wilson, 1988), Templeton (T; 1983), and Kishino-Hasegawa (KH; 1989) tests. These calculations were also done using PAUP*4.0b2. Clade support was assessed using the bootstrap, employing 200 replicates for ML, 500 replicates for ME and MP, and 10,000 puzzling steps for QP. Trees were rooted using marsupial outgroups. Molecular clock estimates of splitting events. The relative evolutionary rate homogeneity between taxa was tested using the method proposed by Wu and Li
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TABLE 1 Accession Numbers and Taxa Common Names Species
Common name
vWF Accession No.
12S-16S Accession No.
Didelphis virginiana Macropus giganteus Bradypus tridactylus Chaetophractus villosus Erinaceus europaeus Scalopus aquaticus Tupaia (glis/tana) Cynocephalus variegatus Megaderma lyra Tadarida brasiliensis Cynopterus sphinx Dobsonia moluccensis Otolemur crassicaudatus Homo sapiens Felis catus Canis familiaris Phocoena phocoena Balaenoptera physalus Sus scrofa Bos taurus Equus (asinus/caballus) Ceratotherium simum Manis sp. Mus domesticus Cavia porcellus Dasyprocta agouti Oryctolagus cuniculus Ochotona princeps Dugong dugon Loxodonta africana Elephas maximus Procavia capensis Orycteropus afer Elephantulus rufescens Amblysomus hottentotus Tenrec ecaudatus Echinops telfairi Micropotamogale lamottei
Opossum Kangaroo Sloth Armadillo Hedgehog Mole Tree shrew Flying lemur Microbat Microbat Megabat Megabat Galago Human Cat Dog Porpoise Fin whale Pig Cow Horse Rhino Pangolin Mouse Guinea pig Agouti Rabbit Pika Dugong African elephant Asian elephant Hyrax Aardvark Elephant shrew Golden mole Tailless tenrec Lesser hedgehog tenrec Nimba otter shrew
AF226848 AJ224670 U31603 AF076480 U97536 AF076479 U31623, AF061063 U31606 U31616 U31623, AF061061 U31605 U31609 U31614, AFO61064 M25851 U31613, AFO61O62 L76227 AF061060 — S78431 X63820, AF004285 U31610 U31604 U97535 U27810 — U31607 U31618 AJ224672 U31608 U31615 U31611 U31619 U31617 U31612 U97534 AF390536* AF076478 AF390538
Z29573 AF027985 AF069535, AF038022 U61080, AF069534 X88898 AF069539 AF038021, AF203727 AF038018, AF038018 AF069538 AF179288 U93068, AF203740 U93065, AF179290 AF019080 J01415 U20753 U96639 — X61145 AJ002189 J01393 X79547 Y07726 U97340, U61079 J01420 L35585 — AJ001588 AF390540* U60185, AF179291 U60182, AF039436 AF390541* U97335, U60184 U97338 U97339 U97336 AF390537* AF069540 AF390539*
Note. Afrotheria members appear underlined.
(1985), as developed in the program K2WuLi-1.0 (Jermiin, 1996), and by the Unambiguous Parsimony Sites test (e.g., Mindell and Honeycut, 1990; Waddell et al., 1999). Taxa showing rate heterogeneity were removed from the analysis (taxa retained for each of the loci were as follows: Vwf: Macropus, Bos, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops; 12S: Macropus, Manis, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong; 16S: Cyclopes, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong; 12S–16S: Balaenoptera, Loxodonta, Elephas, Procavia, Orycteropus, Elephantulus, Micropotamogale, Echinops, Dugong). The resulting pruned taxon set was then analyzed by ML (TN model with parameters estimated as above) under a clock constraint.
We used basal divergences among paenungulate orders (i.e., a hyrax– elephant split, chosen because vWF dugong was not clocklike) at 60 million years ago (MYA) (Amrine and Springer, 1999) as a calibration point for estimating cladogenic events within Tenrecidae. An estimate of variation on these dates was obtained by calculating the split using four different data sets: vWF, 12S rRNA, 16S rRNA, and a concatenation of 12S and 16S rRNA. RESULTS AND DISCUSSION Tenrecidae Monophyly Both the 12S–16S rRNA and the vWF data sets resulted in high support for the monophyly of Tenrecidae (Fig. 1). For the vWF locus, support values (boot-
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FIG. 1. Maximum-likelihood trees based on vWF (A) and 12S–16S (B) data sets. Topologies and branch lengths were determined under a TN model assuming rate variation across sites and employing a proportion of invariant sites.
strap and puzzle) for this clade ranged from 77 to 100%, with only the amino acid puzzle support value below 94% (Table 2). For 12S–16S rRNA, bootstrap support ranged from 80 to 95% (Table 2). Combining the two sets of data into a single concatenated alignment resulted in 100% bootstrap support for the monophyly of Tenrecidae. With the exception of KH-ML for vWF, statistical tests involving a single locus did not provide significant support for Tenrecidae monophyly (Table 2). However, the concatenated data set did provide significant support for tenrecid monophyly, both with MP and ML (Table 2). Asher’s (1999) hypothesis of a paraphyletic Tenrecidae, wherein tenrecines are more closely related to golden moles than to other tenrecids, was rejected with the vWF and concatenated data sets (Table 2). Although statistical tests with the 12S–16S rRNA data set did not reject the Asher hypothesis, bootstrap support was minimal and ranged from 3 to 10%. All of Asher’s (1999) analyses recovered a sister
group relationship between Limnogale and potamogalines, which implies paraphyly of Malagasy tenrecs. This conclusion was mainly supported by a close Potamogalinae–Limnogale (subfamily Oryzorictinae) relationship, but also by an association of the remaining Oryzorictinae with this clade to the exclusion of tenrecines. The recent publication of a 12S rRNA gene sequence for Oryzorictes (Emerson et al., 1999) allowed us to examine the naturalness of a Potamogalinae– Oryzorictinae grouping. After addition of this taxon to our 12S sequences (same selection of taxa as the 12S– 16S analyses), MP, ME, and ML 12S rRNA bootstrap trees supported the monophyly of Tenrecidae (bootstrap support ranged from 47 to 71%) and an Oryzorictes– Tenrecinae clade (bootstrap support ranged from 65 to 80%). Data sets of 12S rRNA sequences confined to afrotherian species (Table 1), with two different sets of outgroups (two Xenarthra or two Chiroptera), recovered additional support for this Oryzorictes– Tenrecinae clade (Table 3). All methods of phylogenetic
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TABLE 2 Tenrecidae Monophyly versus Asher’s Paraphyletic (Tenrecinae ⴙ Chrysochloridae) Hypothesis Tenrecinae ⫹ Golden mole
Tenrecidae
Bootstrap DNA ME (GTR) (Logdet) MP ML (TN) ML (TN GI) Protein MP QP (Blosum 62) QP (Blosum 62 GI) Decay and statistical tests DNA MP Decay KH T WS ML (TN) KH ML (TN GI) KH Protein MP Decay KH T WS
12S–16S
vWF
Concat.
12S–16S
vWF
Concat.
94 95 80 85 93
100 100 99 94 95
100 100 100 100 100
5 3 8 10 6
0 0 0 1 0
0 0 0 0 0
NA NA NA
99 77 77
NA NA NA
NA NA NA
0 ⬍50 ⬍50
NA NA NA
8 0.588–0.634 0.495–0.520 0.402–0.545 0.399 0.204
19 0.189 0.223 0.292 0.000 0.290
31 0.026 0.025 0.049 0.000 0.019
⫺8 0.588–0.634 0.495–0.520 0.402–0.545 0.509 0.538
⫺22 0.046–0.050 0.049–0.052 0.024–0.032 0.005 0.015
⫺38 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.000 0.019
NA NA NA NA
9 0.061 0.061 0.093
NA NA NA NA
NA NA NA NA
⫺13 0.007–0.028 0.007–0.028 0.011–0.043
NA NA NA NA
Note. Statistical test results for Tenrecidae correspond to acceptance of that hypothesis, whereas test results for Tenrecinae ⫹ Golden mole, refer to rejection.
reconstruction, regardless of the root, recovered this association with bootstrap support ranging from 86 to 100%. Bootstrap support for Oryzorictes–Micropotamogale was ⬍1% in all analyses and a subset of statistical tests rejected this hypothesis (Table 3).
Timing and Biogeographic Events The basal split between Potamogalinae and Tenrecinae was estimated at 53, 52, 53, and 51 MYA with vWF, 12S–16S, 12S, and 16S, respectively. Estimates
TABLE 3 Malagasy Tenrecid Monophyly versus Asher’s Paraphyletic (Potamogalinae–Oryzorictinae) Hypothesis Oryzorictes ⫹ Tenrecinae
Bootstrap ME (GTR) (Logdet) MP ML (TN) ML (TN GI) Decay and statistical tests MP Decay KH T WS ML (TN) ML (TN GI)
Oryzorictes ⫹ Micropotamogale
Xenarthra
Chiroptera
Xenarthra
Chiroptera
96 96 86 99 99
97 98 91 99 100
0 0 0 0 0
0 0 0 0 0
⫹6 0.4605 0.4602 0.2076 0.2665 0.0371
⫹6 0.2280 0.2278 0.2913 0.0548 0.0995
⫺9 0.2078 0.6143 0.2626 0.1314 0.0379
⫺12 0.0072–0.0640 0.0073–0.0641 0.0118–0.0896 0.0340 0.0995
Note. Statistical test results for Oryzorictes ⫹ Tenrecinae correspond to acceptance of that hypothesis, whereas test results for Oryzorictes ⫹ Micropotamogale, refer to rejection.
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for the Tenrec–Echinops split within Tenrecinae were more variable (18, 38, 30, 44 MYA). Adding Oryzorictes to the 12S data set resulted in a similar estimate for the split between Potamogalinae and other tenrecs at 55 MYA and placed the Oryzorictinae–Tenrecinae split at approximately 37 MYA. These dates are much older than the fossil record would suggest (i.e., the oldest tenrecids are Miocene; McKenna and Bell, 1997). The separation of Madagascar from mainland Africa occurred between 120 and 165 MYA (Rabinowitz et al., 1983), well before the origin of tenrecids. One or more dispersal events between Africa and Madagascar are thus required to explain the biogeographic history of tenrecids. Given that molecular data are compatible with both tenrecid monophyly and the monophyly of Malagasy tenrecs, the minimal requirement is a single dispersal event from Africa to Madagascar. Further, this event would have occurred after the potamogaline–Malagasy tenrec divergence (51 to 55 MYA) but before the radiation of Malagasy tenrecs (37 MYA based on the 12S rRNA data set that includes tenrecine and oryorictine representation). The mechanism by which tenrecids arrived in Madagascar remains unclear. McCall (1997) argued for a land bridge across the Mozambique Channel. According to this hypothesis, uplift along the Davie fracture zone created a filter bridge by which some terrestrial mammals were able to cross a deep channel with difficult currents, some time between the mid Eocene and the late Oligocene (45 to 26 MYA). The duration of the land bridge thus exhibits partial temporal overlap with the tenrecid dispersal window suggested by our molecular data. Although molecular phylogenies only require a single dispersal event for living tenrecids, fossil data suggest the possibility of a more complex dispersal history. Specifically, the subfamily Geogalinae includes the living Geogale (Madagascar) and the extinct genus Parageogale from the early Miocene of Africa (McKenna and Bell, 1997). Butler (1985) proposed a sister taxon relationship between Geogale and Parageogale. If these taxa are indeed close relatives, then a second dispersal event is required to account for the biogeographic history of the Geogalinae. Depending on the relationship of Geogalinae to other tenrecids, the second dispersal may have been from Africa to Madagascar or vice versa. At present, molecular data do not address this hypothesis. ACKNOWLEDGMENTS Otter shrew (Micropotamogale lamottei) biological material was a generous gift from Peter Vogel (IZEA—University of Lausanne, Switzerland), from a wild-caught specimen from Ye´ ale´ , Ivory Coast (voucher IZEA-7083). Peter Vogel also provided useful comments on an earlier version of the manuscript. This work was supported by grants from the Training and Mobility of Researchers program of the European Commission (ERB-FMRX-CT98-0221) to M.J.S. and the National Science Foundation (DEB-9903810) to M.S.S.
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